Triple helix (“triplex”) structures were first reported in 1957 from the combination of poly-adenylic acid (“poly-A”) with two equivalents of poly-uridylic acid (“poly-U”) (Felsenfeld et al., J. Amer. Chem. Soc. 79: 2023, 1957). It is known that the third pyrimidine strand, which resides in the wider major groove of duplex DNA, recognizes homopurine stretches and binds parallel to the purine strand (referred to as “parallel motif” or “pyrimidine motif”). In another approach, which uses purines in the third strand, the recognition of the purine stretch in the duplex is anti-parallel (referred to as “purine motif” or “anti-parallel motif”).
The general requirement for homogeneous runs of purine/pyrimidine nucleotide bases in the formation of a traditional triple helix structure has resulted from the need to use natural nucleotide bases in the complementary third strand, due to the unavailability of any other molecules to substitute effectively for these natural bases. Traditional third strand binding has therefore been restricted to homogeneous runs of natural purines or pyrimidines because of spacial restrictions associated with Hoogsteen base pairing of the N7— and X6—positions of naturally occurring purines (X is the NH2 or oxygen for adenine and guanine, respectively) in the homopurine strand of the Watson-Crick duplex DNA. Because only the homopurine strand of the duplex provides hydrogen bonding information in such a structure, the third strand binds asymmetrically in the major groove nearest to the sugar-phosphate backbone of the purine strand. As a result, any deviation from homopurine sequence requires that the traditional third strand actually cross over to the other side of the major groove. Limitations in the span and flexibility of the 5′-3′-linked deoxyribose/phosphodiester backbone do not allow this to occur. Thus, any pyrimidine interruption in the homopurine strand cannot be accommodated by the traditional third strand and also significantly destabilizes traditional triple helix formation. In addition to the crossover barrier, the major groove hydrogen-bonding information on the purine molecule targeted by the third strand is not the same for A—T as compared to T—A pairing.
There is intense interest in the design of molecules that can bind sequence specifically via a triple helix motif to mixed purine/pyrimidine sequences in native Watson-Crick DNA (Griffin and Dervan (1989) Science, 245:967-970; Horne and Dervan (1990) J. Am. Chem. Soc., 112:2435-2437; Jayasena and Johnston (1992) Nucl. Acids Res., 20:5279-5288; Gowers and Fox (1999) Nucl. Acids Res., 27:1569-1577; Buchini et al. (2004) J. Angew. Chem. Int. Ed., 43:3925-3928; Craynest et al. (2004) Tetrahedron Lett., 45:6243-6247). To achieve this goal, a set of four C-glycoside bases (Li et al. (2003) J. Am. Chem. Soc., 125:2084-2093), i.e., 2-amino-4-(2′-deoxy-β-D-ribofuranosyl) quinoline (antiGC), 2-amino-5-(2′-deoxy-β-D-ribofuranosyl)quinoline (antiCG), 2-amino-4-(2′-deoxy-β-D-ribofuranosyl)quinazoline (antiAT), and 2-amino-5-(2′-deoxy-β-D-ribofuranosyl) quinazoline (antiTA), which differentiate between the four base paring schemes in the major groove, i.e., G:C, C:G, A:T, T:A, respectively, have been previously generated by the present inventor.
In stark contrast to the traditional triple helix motifs which require homogenous stretches of either purine or pyrimidine nucleotide bases as targets for binding, the above synthetic bases and nucleotides permit any known duplex DNA and/or RNA sequences to be targeted, including the usual duplex DNA and/or RNA sequences which contain heterogeneous (mixed) sequences of purines and pyrimidines. Synthetic oligomers containing these bases recognize major-groove hydrogen bonding information associated with the purine and, optionally, the pyrimidine bases contained in each interstrand nucleotide base-pair combination in the targeted gene sequence. The orientation of the synthetic oligonucleotide relative to the duplex may be arbitrarily defined as running antiparallel to the left strand in the major groove that runs 5′ to 3′ top to bottom. Oligomers comprising the synthetic monomeric compounds described above can form stable sequence-specific triple helix structures with duplex (double-stranded) Watson-Crick DNA molecules, and do so in such a way that the sugar-phosphate backbone of the synthetic oligomer lies near the center of the major groove of the duplex DNA structure. Because these oligomers recognize nucleotide base sequences in double-stranded DNA without the limitation that the binding be done at low pH, or that the targeted sequence be only a homogeneous sequence of either purines or pyrimidines, the construction of triple helix-forming oligomers directed against any known heterogeneous sequence of purines and pyrimidines (as is commonly found in viral or non-viral sequences) is straightforward.
U.S. Pat. No. 5,844,110, which is currently owned by the present applicants, discloses novel monomeric compositions which are substituted quinoline- or quinazoline-based structures capable of hydrogen bonding specifically with interstrand purine-pyrimidine base pairs in a double-stranded Watson-Crick DNA molecule. The monomeric compounds of the '110 patent are capable of being assembled in specific sequences into oligomers capable of binding with sequence specificity to duplex DNA via a triple helix motif.
Of the four C-glycoside bases described and claimed in the '110 patent, antiTA (Li et al. (2003) J. Am. Chem. Soc., 125:2084-2093), antiGC (Li et al. (2004) Biochemistry, 43:1440-1448), and antiCG (Li et al. (2005) submitted J. Am. Chem. Soc.) have been synthesized by the coupling of a protected ribofuranoid glycal with a halogenated heterocycle using a Pd-mediated Heck-type reaction (Cheng et al. (1985) J. Org. Chem., 50:2778-2780; Davies, G. D. (1992) J. Org. Chem., 57:4690-4696; Farr et al. (1992) J. Org. Chem., 57:2093-2100; Farr et al. (1990) Carbohydr. Chem., 9:653-660). Notably, the synthesis of oligomers with antiAT by solid phase synthesis has proven to be less than ideal because of partial decomposition of antiAT during deprotection unless prolonged deprotection times at lower temperatures were employed.